International Journal of
Molecular Sciences
Article
Peculiarities of the Super-Folder GFP Folding
in a Crowded Milieu
Olesya V. Stepanenko 1 , Olga V. Stepanenko 1 , Irina M. Kuznetsova 1 , Vladimir N. Uversky 1,3, *
and Konstantin K. Turoverov 1,2, *
1
2
3
*
Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology,
Russian Academy of Sciences, 4 Tikhoretsky Ave., St. Petersburg 194064, Russia; [email protected] (Ole.V.S.);
[email protected] (Olg.V.S.); [email protected] (I.M.K.)
Institute of Physics, Nanotechnology and Telecommunications, Peter the Great St. Petersburg State
Polytechnic University, 29 Polytechnicheskaya st., St. Petersburg 195251, Russia
Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute,
Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC07,
Tampa, FL 33612, USA
Correspondence: [email protected] (V.N.U.); [email protected] (K.K.T.);
Tel.: +1-813-974-5816 (V.N.U.); +7-812-297-1957 (K.K.T.); Fax: +1-813-974-7357 (V.N.U.); +7-812-297-0341 (K.K.T.)
Academic Editor: Salvador Ventura
Received: 26 August 2016; Accepted: 20 October 2016; Published: 28 October 2016
Abstract: The natural cellular milieu is crowded by large quantities of various biological
macromolecules. This complex environment is characterized by a limited amount of unoccupied
space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly
packed cellular environment is poorly mimicked by traditional physiological conditions, where low
concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative
is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution
containing high concentrations of various polymers that serve as model crowding agents. An expected
outcome of the presence of such macromolecular crowding agents is their ability to increase
conformational stability of a globular protein due to the excluded volume effects. In line with
this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and
concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas
the chemical nature of a macromolecular crowder should not play a role in its ability to modulate
conformational properties. In this study, the effects of different crowding agents (polyethylene glycols
(PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70)
on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent
protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that,
in addition to the expected excluded volume effects, there are some changes in the solvent properties.
Keywords: super-folder GFP; protein folding; conformational stability; crowded milieu; excluded
volume effect; solvent properties
1. Introduction
Fluorescent protein markers (FPs) have become a valuable tool for solving many problems
of molecular and cell biology using fluorescence microscopy [1–7]. The green fluorescent protein
(GFP)-like fluorescent proteins have a special place among them, as they are relatively small proteins
with a β-barrel structure containing a unique chromophore that absorbs and emits in the visible spectral
region [8]. Compared to the well-known enhanced green fluorescent protein (EGFP), the amino acid
sequence of a super-folder GFP (sfGFP) has nine amino acid substitutions. This protein does not
have the drawbacks of other FPs, such as the tendency to aggregate and the irreversibility of their
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denaturation [9–12]. sfGFP is characterized by high efficiency of its folding, as it is able to fold even
being fused to a poorly-folded partner peptide [10]. Furthermore, sfGFP in such a fusion construct
increases the folding efficiency of its partner that it can be used for the expression of poorly-soluble
recombinant proteins and peptides [13,14]. These properties of sfGFP defined the use of this protein as
a convenient model subject for the folding-unfolding study of FPs.
We have previously investigated the equilibrium unfolding-folding processes of sfGFP induced
by guanidine thiocyanate (GTC) in vitro [15,16]. The GTC-induced unfolding of sfGFP was shown
to be reversible. This was confirmed by the complete recovery of all characteristics recorded for
the pre-denatured sfGFP after refolding of this protein from its completely unfolded state. During
the protein refolding, an off-pathway intermediate state was shown to be formed in the presence
of the moderate denaturant concentrations. In this intermediate state, the β-barrel structure was
almost completely formed, but the inner α-helix bearing the chromophore and a single tryptophan
residue of the protein (Trp57) were not properly incorporated into the protein matrix. As for other
FPs analyzed so far, the unfolding-refolding processes of sfGFP was characterized by hysteresis.
The hysteretic behavior of FPs at their unfolding-refolding process is determined by the presence of the
chromophore [12,16–19]. Indeed, folding of sfGFP in native conditions (after the protein biosynthesis)
occurs in the absence of any chromophore (which happens at the latter maturation stage), whereas the
protein undergoing refolding from the fully unfolded state already bears the mature chromophore
(which, being a covalent adduct, is not destroyed by high concentrations of strong denaturants).
Ones being formed, the chromophore strongly contributes not only to the protein refolding processes,
but also to the protein conformational stability, by holding a compact structure of the β-barrel through
non-covalent interactions with the surrounding protein matrix [15]. Notably, while studying the
structural perturbations of FPs, and sfGFP in particular, the chromophore serves as a convenient and
reliable marker of protein structural integrity, since it has a high quantum yield, only being properly
incorporated into the β-barrel [3,16,20–22].
An interesting feature of FPs is a specific change in the spectral properties of the protein
chromophore in the presence of ionic denaturants and salts [15,23], which should be considered
in the study of the folding of these proteins. In fact, these constituents (ionic denaturants and salts)
expressed strong influence on the spectral characteristics of the chromophore of sfGFP, without
inducing disruption of the integrity of the protein structure [15,23]. The binding of an anion of the
salt or ionic denaturant near the chromophore shifts the equilibrium between the neutral and the
anionic forms of the chromophore and is not associated with the appearance of any partially-folded
intermediates during the protein unfolding.
Since FPs are widely used as markers of various cellular processes, the study of the structural
and spectral properties of these proteins under conditions simulating those found in the cellular
environment; i.e., under macromolecular crowding, is of great interest. This crowded environment
is characterized by the limited space available for a protein [24,25] and by the restricted amount
of free water [24,26]. These environmental perturbations significantly influence a variety of
biologically-relevant processes, such as the folding process, the binding of small molecules, enzymatic
activity, protein-protein interactions, amyloid formation, etc. [24,27–32]. In laboratory practice,
different polymer molecules, such as polyethylene glycol (PEG) of different molecular weights,
Dextran, Ficoll, and inert proteins, are typically used as crowding agents [25,28,29,31]. We, and others,
established that the excluded volume effect is not the only factor influencing the behavior of the protein
in a crowded milieu [29,33,34]. Apparently, the special role belongs to the ability of the crowding
agents to alter the properties of the solvent. This conclusion is based on the analysis of the urea-induced
unfolding behavior of globular proteins belonging to different structural classes and having different
physicochemical properties in the presence of the two commonly used crowding agents, PEG-8000
and Dextran-70. Particularly, our studies showed that globular proteins can be divided into the
three categories: highly-stable proteins with no influence of the crowding agents, proteins markedly
stabilized by polymers, and proteins appreciably destabilized by at least one of the crowding agents.
Int. J. Mol. Sci. 2016, 17, 1805
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Therefore, different polymers carry very different effects on the conformational stability of a protein.
In the study of the folding-unfolding processes of bovine odorant-binding protein in the crowded
conditions it was found out that the effect of the crowding agent PEG on the native protein structure,
and its stability, strictly depends on the molecular weight of this crowder [35].
In the present study, we investigated the effect of different crowding agents on the spectral
properties and unfolding-refolding processes of sfGFP induced by the GTC. PEGs of various molecular
masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70, were used as crowding agents
in this study.
2. Results
2.1. Spectral Properties of sfGFP in the Presence of Crowding Agents
The impact of crowding agents on the structure and spectral properties of sfGFP was studied first.
To this end, we recorded the absorption spectra in the UV and visible region (i.e., the absorption
originating from the tryptophan residue and the chromophore of the protein), the tryptophan
fluorescence spectra and the fluorescence of the green chromophore (at λex = 390 nm), the fluorescence
excitation spectra of the chromophore (at λreg = 540 nm), the CD spectra in the far- and near-UV
and visible regions. PEGs of different molecular weights (600, 8000, and 12,000 Da), Dextran-70,
and Ficoll-70 were used as crowding agents. The action of crowding agents in low, medium, and high
concentrations was tested.
The absorbance of protein samples did not exceed 0.15 in all of our experiments unless otherwise
indicated. Taking into account the extinction coefficient ε280 = 31,519 M−1 ·sm−1 [19], this means
that protein concentration was not higher than 5 µM. Therefore, we assumed that sfGFP existed
as a monomer at our experimental conditions (low protein concentrations were needed for the
spectroscopic characterization). It is worth noting that the monomerization of avGFP requires the
disruption of the hydrophobic surface of the protein at the dimeric interface by introducing a set
of charged residues in the key positions 206, 221, and 223. The sfGFP has hydrophobic residues
Val 206, Leu 221, and Phe 223 at these positions (PDB file 2B3P [10]). This may be a reason for the
sfGFP dimerization, especially at the conditions that favor aggregation, such as macromolecular
crowding conditions. However, at the protein concentrations used in our studies, the aforementioned
hydrophobic surface of sfGFP does not prevent a complete protein refolding in the absence of crowding
agents [15]. Furthermore, it was shown that sfGFP behaves as a monomer in the cell [36].
We found no significant changes in the spectral characteristics of sfGFP in the presence of
Dextran-70 (see Supplementary Materials, Figure S1) and Ficoll-70 (see Supplementary Materials,
Figure S2), PEG-600 (Figure S3), and PEG-8000 (Figure S4) at all tested concentrations of the crowding
agents and in the presence of PEG-12000 at concentrations below 20% (Figure 1). Some reduction
in the intensity of the UV-absorption band of sfGFP can be observed in the presence of Ficoll-70
(Figure S2). This is probably due to the fact that the crowding agent absorbs strongly in this spectral
region (data not shown). It should be noted that the absorption spectra of sfGFP in the presence of
30% of PEG-8000 and of 20%–30% of PEG-12000 demonstrate a marked increase in light scattering.
The strong increase in light scattering for the protein solution in the presence of 30% of PEG-12000
led to a significant distortion of the absorption spectrum of the protein. The protein solutions at these
conditions were opalescent.
In addition to the increase in light scattering, an evident change in the spectral properties of
sfGFP in the presence of 30% of PEG-12000 can be seen (Figure 1). At these conditions, there was
some observed increase in the intensity of the shoulder at 390 nm and a slight redshift of the peak at
490 nm in the absorption, excitation, and CD spectra in the visible region of sfGFP. The far-UV CD
spectra and the tryptophan fluorescence spectra of sfGFP were significantly distorted in the presence
of high concentrations of PEG-12000. The decrease in the intensity of absorption and fluorescence of
the tryptophan residue and the chromophore of sfGFP can also be observed. These effects become
Int. J. Mol. Sci. 2016, 17, 1805
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even more pronounced with the two-fold increase in the protein concentration (data not shown).
Therefore,the
theaforementioned
aforementionedspectral
spectral changes of
bebe
attributed
to to
Therefore,
of sfGFP
sfGFPunder
underthese
theseconditions
conditionscan
can
attributed
the
aggregation
of
protein
molecules.
the aggregation of protein molecules.
Figure1.1.The
Theeffect
effect of
of crowding
crowding agent PEG-12000
Figure
PEG-12000on
onthe
thespectral
spectralproperties
propertiesofofsfGFP.
sfGFP.Absorption
Absorption
spectra(A);
(A);tryptophan
tryptophanfluorescence
fluorescencespectra
spectra at
at λex
ex ==295
spectra
of of
thethe
protein
spectra
295nm
nmand
andthe
thefluorescence
fluorescence
spectra
protein
ex =
spectra
in the
far far
(the(the
datadata
are represented
in units
the of
chromophoreat
at λλex
chromophore
= 390
390nm
nm(B);
(B);and
andCD
CD
spectra
in the
are represented
in of
units
molar
ellipticity
per per
amino
acid acid
residue),
near (in
units
the molar
and visible
regionUV
the
molar
ellipticity
amino
residue),
near
(inof
units
of theellipticity),
molar ellipticity),
andUV
visible
(in units
the ellipticity)
were recorded
(C). The applied
of PEG-12000
8% (green
region
(inof
units
of the ellipticity)
were recorded
(C). Theconcentrations
applied concentrations
of were
PEG-12000
were
line),
20%
(gray
line),
and
30%
(red
line).
The
spectra
of
sfGFP
in
the
buffered
solution
(black
lines)
8% (green line), 20% (gray line), and 30% (red line). The spectra of sfGFP in the buffered solution
are shown
(black
lines) for
arecomparison.
shown for comparison.
The protein aggregation probably leads to shielding and scattering of the light beam, which
The protein aggregation probably leads to shielding and scattering of the light beam, which
resulted in a decrease in the proportion of light transmitted through the protein solution. This may
resulted
a decrease
in and
the proportion
transmitted
thelight
protein
solution.
decreaseinthe
absorption
fluorescenceofoflight
a sample.
As it is through
known, the
scattering
can This
lead may
to
decrease
the
absorption
and
fluorescence
of
a
sample.
As
it
is
known,
the
light
scattering
lead
a reduced CD signal. In our case, the strong light scattering resulted in the distortion of the CD can
signal
toofa sfGFP.
reduced
CDdeformation
signal. In our
case, the
strong
light scattering
resulted in
distortion
of theofCD
The
of sfGFP
β-barrel
induced
by its aggregation
canthe
affect
the network
signal
of sfGFP. The
deformation
of sfGFP the
β-barrel
induced and
by its
affect
the network
intramolecular
hydrogen
bonds between
chromophore
theaggregation
side chainscan
of the
residues
that
ofform
intramolecular
hydrogen
bonds
between
the
chromophore
and
the
side
chains
of
the
residues
the β-barrel of sfGFP. This, in turn, may shift the equilibrium between protein molecules
that
form theneutral
β-barrel
sfGFP.forms
This, of
in the
turn,
may shift the
equilibrium
protein
containing
andofanionic
chromophore
[23],
resulting inbetween
an increase
of themolecules
peak at
containing
and anionic
formsand
of the
[23], resulting
anredshift
increase
peak
390 nm in neutral
the absorption,
excitation,
thechromophore
visible CD spectra
of sfGFP. in
The
ofof
thethe
peak
at at
390
and the
visible
of sfGFP.
redshift
of the peak
490nm
nmininthe
theabsorption,
absorption excitation,
spectra of sfGFP
testifies
forCD
thespectra
stabilizing
of theThe
anionic
chromophore
of at
thenm
protein
490
in the[23,37,38].
absorption spectra of sfGFP testifies for the stabilizing of the anionic chromophore of the
protein [23,37,38].
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2.2. Unfolding-Refolding of sfGFP in the Crowded Milieu
2.2. Unfolding-Refolding of sfGFP in the Crowded Milieu
While studying the processes of sfGFP folding-unfolding induced by GTC in the absence of
crowding
we the
haveprocesses
shown that
the disruption
of the protein
structure
occurs
the 0.9–
Whileagents,
studying
of sfGFP
folding-unfolding
induced
by GTC
in within
the absence
of
1.7
M GTCagents,
range (Figure
2, black
Observed
changesstructure
of all recorded
crowding
we have
shownlines
thatand
thesymbols).
disruption
of the protein
occurs parameters
within the
of
sfGFP
a GTC
concentration
below
0.7and
M are
caused by
the binding
of aof
negatively-charged
SCN−
0.9–1.7
MatGTC
range
(Figure 2, black
lines
symbols).
Observed
changes
all recorded parameters
ions
in the
of the chromophore
with
redistribution
of the protein molecules
of sfGFP
at avicinity
GTC concentration
below 0.7 M
aresubsequent
caused by the
binding of a negatively-charged
SCN−
bearing
thevicinity
chromophore
in the anionic
and
neutral forms,
the inhibition
of proton
transferbearing
in the
ions in the
of the chromophore
with
subsequent
redistribution
of the protein
molecules
excited
state of the
the quenching
the chromophore
fluorescence
the sulfur
the chromophore
inchromophore,
the anionic andand
neutral
forms, the of
inhibition
of proton transfer
in theby
excited
state
atom
SCN− ions [15,23].
of theof
chromophore,
and the quenching of the chromophore fluorescence by the sulfur atom of SCN−
Here, we have registered the dependencies of the different spectral characteristics of sfGFP
ions [15,23].
during
the GTC-induced
unfolding-refolding
processes
the presence
of different
crowding
Here,
we have registered
the dependencies
of theindifferent
spectral
characteristics
of agents
sfGFP
of
different
concentrations
to test effect of crowding
agents
inpresence
a wide range
of sizescrowding
on sfGFPagents
stability
during
the GTC-induced
unfolding-refolding
processes
in the
of different
of
and
folding.
The
experimental
data
in
the
presence
of
PEGs
of
different
molecular
weights
are
different concentrations to test effect of crowding agents in a wide range of sizes on sfGFP stability and
presented
inexperimental
the Figures 2–4,
forpresence
the Dextran-70
Ficoll-70molecular
are shownweights
in the Figures
5 and in
6,
folding. The
dataand
in the
of PEGsand
of different
are presented
respectively.
the Figures 2–4, and for the Dextran-70 and Ficoll-70 are shown in the Figures 5 and 6, respectively.
Figure 2. The
onon
thethe
sfGFP
conformational
changes
induced
by
Theeffect
effectofofcrowding
crowdingagent
agentPEG-12000
PEG-12000
sfGFP
conformational
changes
induced
GTC.
Changes
of of
parameter
AA
(A);
by GTC.
Changes
parameter
(A);light
lightscattering
scattering(B);
(B);corrected
correctedfluorescence
fluorescence intensity
intensity of
of green
chromophore
two
wavelengths
of excitation
390
490 and
nm changes
(D); and
of
chromophore atattwo
wavelengths
of excitation
of 390ofnm
(C)nm
and(C)
490and
nm (D);
of changes
absorbance
absorbance
390490
nmnm
(E)(F)and
nm Open
(F) aresymbols
shown.indicate
Open symbols
indicate
unfolding
filled
at 390 nm (E)atand
are490
shown.
unfolding
and filled
symbolsand
represent
symbols
refolding.
performed of
after
24 or
h denatured
incubation protein
of native
or
refolding.represent
Measurements
were Measurements
performed afterwere
24 h incubation
native
in the
presence
GTC orinGTC
and the crowding
Thethe
applied
concentrations
of PEG-12000
were 8%
denaturedofprotein
the presence
of GTC oragent.
GTC and
crowding
agent. The applied
concentrations
(blue
circles), 20%
(green
and 30%
(red(green
circles).
The unfolding-refolding
curvesThe
of sfGFP
in the
of PEG-12000
were
8% circles),
(blue circles),
20%
circles),
and 30% (red circles).
unfoldingabsence ofcurves
the crowding
also shown
(black circles
and
refolding
of sfGFPagent
in theare
absence
of the crowding
agent
arelines).
also shown (black circles and lines).
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Figure
3.The
The
effect
ofcrowding
crowding
agent
PEG-8000
onthe
thesfGFP
sfGFP
conformational
changes
induced
by
Figure
3. 3.
The
effect
of of
crowding
agent
PEG-8000
onon
the
sfGFP
conformational
changes
induced
by by
GTC.
Figure
effect
agent
PEG-8000
conformational
changes
induced
GTC.
For
moresee
details
seethe
thelegend
Applied
concentrations
PEG-8000
were
8%(blue
(blue
ForGTC.
moreFor
details
the legend
oflegend
Figure
2.Figure
Applied
concentrations
of PEG-8000
were 8%
(blue
circles),
more
details
see
ofofFigure
2.2.Applied
concentrations
ofofPEG-8000
were
8%
circles),
12%
(green
circles),
and30%
30%(red
(redcircles).
circles).
circles),
(green
circles),
and
12%
(green12%
circles),
and
30% (red
circles).
Figure
4. 4.
The
effect
of of
crowding
agent
PEG-600
on on
the
sfGFP
conformational
changes
induced
by by
GTC.
Figure
4.The
The
effect
ofcrowding
crowding
agent
PEG-600
onthe
thesfGFP
sfGFP
conformational
changes
induced
by
Figure
effect
agent
PEG-600
conformational
changes
induced
ForGTC.
moreFor
details
the legend
Figure
Applied
concentrations
of PEG-600
were 8%
(blue
circles),
GTC.
For
moresee
details
seethe
theof
legend
Figure
Applied
concentrations
PEG-600
were
8%(blue
(blue
more
details
see
legend
ofof2.
Figure
2.2.Applied
concentrations
ofofPEG-600
were
8%
circles),
20%
(green
circles),
and30%
30%(red
(redcircles).
circles).
20%
(green20%
circles),
and
30% (red
circles).
circles),
(green
circles),
and
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Mol. Sci.
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77 of
Figure
5. The
The
effect
of crowding
crowding
agentDextran-70
Dextran-70 on
on the
the conformational
conformational
changes
of of
sfGFP
induced
Figure
5. The
effect
of crowding
agent
conformationalchanges
changes
sfGFP
induced
Figure
5.
effect
of
agent
Dextran-70
on
of
sfGFP
induced
by
GTC.
For
more
details
see
the
legend
of
Figure
2.
Applied
concentrations
of
Dextran-70
were
24%24%
by GTC.
ForFor
more
details
seesee
the
Appliedconcentrations
concentrations
Dextran-70
were
by GTC.
more
details
thelegend
legendof
ofFigure
Figure 2.
2. Applied
of of
Dextran-70
were
24%
(green
circles),
and
30%
(red
circles).
(green
circles),
andand
30%
(red
circles).
(green
circles),
30%
(red
circles).
Figure 6. The effect of crowding agent Ficoll-70 on the conformational changes of sfGFP induced by
Figure
6. The
effectofofcrowding
crowding agent
agent Ficoll-70
thethe
conformational
changes
of sfGFP
induced
by
Figure
6. The
effect
Ficoll-70onon
conformational
changes
of sfGFP
induced
GTC. For
For more
more details
details see
see the
the legend
legend of
of Figure
Figure 2.
2. Applied
Applied concentrations
concentrations of
of Ficoll-70
Ficoll-70 were
were 8%
8% (blue
(blue
GTC.
by GTC. For more details see the legend of Figure 2. Applied concentrations of Ficoll-70 were 8%
circles), 20%
20% (green
(green circles),
circles), and
and 30%
30% (red
(red circles).
circles).
(bluecircles),
circles), 20%
(green circles),
and 30%
(red circles).
Denaturation dependencies of all registered characteristics of sfGFP in the presence of all tested
concentrations of Dextran-70 (Figure 5) and Ficoll-70 (Figure 6) coincided with the unfolding curves of
sfGFP in the absence of crowding agents.
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Denaturation dependencies of all registered characteristics of sfGFP in the presence of all tested
Int. J. Mol. Sci. 2016, 17, 1805
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concentrations of Dextran-70 (Figure 5) and Ficoll-70 (Figure 6) coincided with the unfolding curves
of sfGFP in the absence of crowding agents.
However, PEGs of various molecular weights differently affected the
the unfolding-refolding
unfolding-refolding
processes of sfGFP. In
In the
the presence
presence of
of PEG-600
PEG-600 (Figure
(Figure 4)
4) and
and PEG-8000
PEG-8000 (Figure 3), the unfolding
curves of sfGFP were shifted to a higher denaturant concentrations compared to sfGFP denaturation
solution
without
crowding
agents.agents.
Furthermore,
this shiftthis
toward
higher
denaturant
curves ininbuffered
buffered
solution
without
crowding
Furthermore,
shift
toward
higher
concentrations
elevated
with
the
increase
in
the
concentration
of
crowding
agent.
Therefore,
PEG-600
denaturant concentrations elevated with
increase in the concentration of crowding agent.
and PEG-8000
had concentration-dependent
stabilizing effects onstabilizing
the structure
of sfGFP.
Therefore,
PEG-600
and PEG-8000 had concentration-dependent
effects
on the Contrary,
structure
PEG-12000
had both aPEG-12000
destabilizing
a stabilizing
effect on
The addition
this
crowding
of
sfGFP. Contrary,
hadand
both
a destabilizing
andsfGFP.
a stabilizing
effectofon
sfGFP.
The
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(8%,inFigure
was followed
by the
shift2)ofwas
sfGFP
denaturation
curves
addition
thisconcentration
crowding agent
a low 2)
concentration
(8%,
Figure
followed
by the shift
of
to lower
concentrations
oftothe
denaturant.
At concentrations
exceeding
20%, similarly
to the
sfGFP
denaturation
curves
lower
concentrations
of the denaturant.
At concentrations
exceeding
PEG-600
and PEG-8000,
PEG-12000
had a stabilizing
effect
onathe
structureeffect
of sfGFP
and
this effect
20%,
similarly
to the PEG-600
and PEG-8000,
PEG-12000
had
stabilizing
on the
structure
of
was
concentration-dependent.
sfGFP
and this effect was concentration-dependent.
In order to compare the impact of PEGs with different molecular weight on the unfolding
sfGFP, the
the denaturation
denaturation curves of the protein in the presence of these agents were put
process of sfGFP,
together in the same plots. Figure
Figure 77 and Figure S5 show that at the concentration of 8%, PEG-600
stabilized
thestructure
structure
of sfGFP
slightly,
the stabilizing
effect of was
PEG-8000
was more
stabilized the
of sfGFP
slightly,
while while
the stabilizing
effect of PEG-8000
more pronounced,
pronounced,
and
PEG-12000
had
a noticeableeffect
destabilizing
effect
on the sfGFP
When the
and PEG-12000
had
a noticeable
destabilizing
on the sfGFP
structure.
Whenstructure.
the concentration
of
concentration
of crowding
agents
were
30%, the
exerted
by PEGs
on thePEG-600
protein
crowding agents
were raised
to 30%,
theraised
impacttoexerted
by impact
PEGs on
the protein
changed.
changed.
PEG-600
had the most
pronounced
stabilizing
effectofon
the structure
sfGFP.
Further,ofwith
had the most
pronounced
stabilizing
effect on
the structure
sfGFP.
Further,of
with
increasing
the
increasing
of the molecular
weight
of the
the extent
of on
its the
stabilizing
effectreduced.
on the
molecular weight
of the crowding
agent,
thecrowding
extent of agent,
its stabilizing
effect
protein was
protein
was reduced.of
At20%,
the concentration
of 20%,
all PEGs
sfGFP, witheffect
a more
pronounced
At
the concentration
all PEGs stabilized
sfGFP,
withstabilized
a more pronounced
being
induced
effect
induced
by with
the crowding
agents with
higher molecular weights.
by thebeing
crowding
agents
higher molecular
weights.
Figure
onon
thethe
denaturation
of sfGFP
induced
by
Figure 7.
7. The
Theeffect
effectofofPEGs
PEGsofofdifferent
differentmolecular
molecularweights
weights
denaturation
of sfGFP
induced
GTC.
The
change
of
parameter
A
(A
=
I
320
/I
365
)
on
GTC
in
the
presence
of
PEG-600,
PEG-8000,
and
by GTC. The change of parameter A (A = I320 /I365 ) on GTC in the presence of PEG-600, PEG-8000,
PEG-12000
(designations
are are
given
in red,
green
and
and PEG-12000
(designations
given
in red,
green
andblue,
blue,respectively).
respectively).The
Theapplied
applied concentrations
concentrations
of
PEGs were
were 8%,
8%, 20%,
20%, and
and 30%
30% presented
presented as
as triangles
triangles (and
(and solid
solid line),
line), circles
circles (and
(and dashed
dashed line)
line) and
and
of PEGs
squares (and dotted line), respectively.
respectively.
With the increase in the molecular weight and concentration of PEGs, an increase in the light
With the increase in the molecular weight and concentration of PEGs, an increase in the light
scattering was observed, indicating the aggregation of the native sfGFP molecules on the pathway of
scattering was observed, indicating the aggregation of the native sfGFP molecules on the pathway of
the protein unfolding, whereas the addition of Dextran-70 or Ficoll-70 did not lead to the aggregation
the protein unfolding, whereas the addition of Dextran-70 or Ficoll-70 did not lead to the aggregation
of native sfGFP molecules in the unfolding process of this protein (Figures 2–6).
of native sfGFP molecules in the unfolding process of this protein (Figures 2–6).
Reversibility of the GTC-induced unfolding of sfGFP was affected in the presence of all crowding
Reversibility of the GTC-induced unfolding of sfGFP was affected in the presence of all crowding
agents used in this study. In fact, the protein failed to recover spectral characteristics recorded for the
agents used in this study. In fact, the protein failed to recover spectral characteristics recorded
native protein prior to its unfolding (Figures 2–6). This indicated the irreversibility of the sfGFP
for the native protein prior to its unfolding (Figures 2–6). This indicated the irreversibility of the
unfolding in the presence of crowding agents. It is worth noting that an increase in the light scattering
sfGFP unfolding in the presence of crowding agents. It is worth noting that an increase in the light
was detected at sfGFP refolding in the presence of all crowding agents, indicating that the protein
scattering was detected at sfGFP refolding in the presence of all crowding agents, indicating that the
refolding process under these conditions is complicated by protein aggregation (Figures 2–6). The
protein refolding process under these conditions is complicated by protein aggregation (Figures 2–6).
The strongest protein aggregation was observed in the presence of PEGs of all molecular weights
Int. J. Mol. Sci. 2016, 17, 1805
9 of 14
(Figures 2–4), whereas the aggregation of sfGFP during refolding was less pronounced in the presence
of Dextran-70 (Figures 5 and 6). Notably, the protein aggregation was stronger at the refolding of
sfGFP compared to the unfolding pathway of this protein.
3. Discussion
The most common explanation of the effects of macromolecular crowding on various physiological
processes is provided by the excluded volume theory. According to this theory, inside the limited
space caused by macromolecular crowding, the equilibrium in the protein folding processes is shifted
to more compact and more folded forms of a protein [24,27–29]. Thus, the macromolecular crowding
favors the formation of the native form of a protein during its folding. It is believed that while
modeling the crowding milieu with different crowding agents, their effects on the protein depend
on the molecular mass of the crowder and its concentration and power with the increase of the
polymer concentration [39]. The efficiency of macromolecular crowding is expected to be highest at
conditions where hydrodynamic dimensions of a crowding agent are comparable to those of a tested
protein [40,41]. In addition to the effect of excluded volume, the presence of a crowding agent in
a solution may affect the properties of a tested protein in some other ways. In the presence of some
direct interactions of a target protein with a crowding agent, the excluded volume effects can be either
strengthened or weakened [42,43].
In this study, PEGs of different molecular weights, together with Ficoll-70 and Dextran-70,
were applied to test the effects of variously-sized crowding agents on the stability and folding of
sfGFP. Here, we tested the crowding agents with sizes ranging from about 6 Å to more than 60 Å.
The Stokes radius of 23.8 Å of native sfGFP is in the middle of this range. The unfolded sfGFP is
a more expanded protein form with a Stokes radius of 48.1 Å. The excluded volume theory suggests
that the crowded environment should promote protein folding through an indirect stabilizing effect
on the folded states of proteins by favoring more compact over less compact species. Within the
frame of this excluded volume theory, this folding promoting effect of crowed media should increase
with the increase in the crowding agent concentration and be more profound for the crowding agent
with the hydrodynamic dimensions similar to those of a query protein. We observed more complex
dependence of sfGFP stability and refolding on the presence of crowding agents. In fact, our previous
data of the analysis of the urea-induced unfolding of structurally-different globular proteins [33,34],
and the results of this study allow the conclusion that even in the absence of direct interaction between
a crowding agent and the target protein the excluded volume theory cannot always be used to explain
the experimental data. In fact, we show here that all crowding agents used in this study do not affect
the structure of sfGFP. In the presence of high concentrations of PEG-12000, the observed changes of
the spectral characteristics of sfGFP are caused by the protein aggregation rather than by a change of
protein’s spatial structure. These data indicate that sfGFP does not interact directly with the crowding
agents used here.
The presence of Ficoll-70 and Dextran-70 do not affect the conformational stability of sfGFP (it does
not lead to the detectable shifts of the unfolding curves registered at the GTC-induced unfolding of
this protein). Notably, the effective hydrodynamic radii of Ficoll-70 and Dextran-70 are 49.3 and 63.9 Å,
respectively (calculated according to the formulas from [44]). The β-barrel of sfGFP is characterized by
a diameter of 24 Å and a height of 42 Å [37], and native GFP (a globular protein with the molecular
mass of 26,886 Da) is expected to have a Stokes radius of 23.8 Å, whereas the completely unfolded
form of this protein is expected to have a Stokes radius of 48.1 Å [45]. Therefore, the lack of the
evident influence of Ficoll-70 and Dextran-70 on the sfGFP unfolding-refolding processes and on
the conformational stability of this protein is in apparent contradiction with the excluded volume
theory, according to which the presence of high concentrations of crowding agents is expected to have
an impact on the structural and conformational properties of a target protein.
The hydrodynamic radii of PEG-600, PEG-8000, and PEG-12000 are 5.6 Å ± 0.3 Å, 24.5 Å ± 1.9 Å,
and 30.9 Å ± 2.5 Å, respectively [28]. According to the excluded volume theory, the greatest effect on
the unfolding-refolding processes should be provided by PEG-8000, whose hydrodynamic dimensions
Int. J. Mol. Sci. 2016, 17, 1805
10 of 14
are closest to those of sfGFP. According to our data, although PEGs influenced the conformational
stability of sfGFP against the denaturing action of GTC, the impact of these crowding agents depended
on the PEG molecular mass and concentration in a complex manner. At the PEG concentration
of 8%, the crowder with the highest molecular weight caused some destabilization of the sfGFP
structure, whereas the other two PEGs increased the resistance of the protein toward the GTC-induced
unfolding. At concentrations of these crowding agents of 20%, their stabilizing effect on the sfGFP
structure increased with the molecular weight of PEG. Finally, at the highest concentration of crowding
agents (30%), PEG of lower molecular weight had the most pronounced stabilizing effect on sfGFP.
The diversity of the effects of PEGs on the stability of sfGFP was not determined by the difference in
interaction of this protein with the crowding agents, since none of the crowders used in our study
caused noticeable changes in the spectral properties of sfGFP.
Our data indicated that during sfGFP refolding in the presence of all crowding agents used in this
study, the recovery of protein characteristics to a level of the native protein was not observed even
under strongly refolding conditions. The increase in the parameter A of the protein is insignificant,
while the recovery in chromophore fluorescence was not detected. Furthermore, in the presence
of all crowding agents, the changes of parameter A of sfGFP during refolding correlates well with
the changes in the light scattering of the protein. In the intermediate state, the structure around the
chromophore of sfGFP is fully recovered, while the environment of the tryptophan residue of sfGFP
slightly differs from that in the native state of this protein [15]. These observations allowed us to
conclude that during sfGFP refolding in the crowded milieu, the native or intermediate protein states
were not formed. The observed changes of sfGFP characteristics can be attributed to the increase in the
rigidity of the tryptophan environment caused by the aggregation of denatured protein molecules.
It is worth mentioning that in avGFP, the hydrogen bond network that involves Ser65 and
Tyr66 residues of the chromophore, a water molecule, and the Ser205 and Glu222 residues from the
chromophore’s close environment is known to play a crucial role in the chromophore maturation and
the FP fluorescent properties’ acquisition [16,46,47]. Reversely, the chromophore strongly contributes
not only to the protein refolding processes, but also to the protein conformational stability, by holding
a compact structure of the β-barrel through the non-covalent interactions with the surrounding protein
matrix [15]. Based on the Fourier transform infrared study on the state of water sorbed to PEG films it
has been concluded that this polymer (which was used as one of the crowding agents in our study)
can not only sorb the water molecules (where water molecules bind to the oxygen atoms of PEG with
one of their hydrogen atoms leading to the formation of “binding water”), but can also affect the
dynamic behavior of water by forming a dimeric water molecule by the association of the free water
molecule with the binding water [48]. Therefore, it is likely that the sorption of the water molecules
by the crowding agent may result in the destruction of the hydrogen network in sfGFP, affecting
conformational stability and refolding of this protein.
4. Materials and Methods
4.1. Materials
GTC (Fluka, Buchs, Switzerland) and crowding agents PEG-600, PEG-8000, and PEG-12000,
Dextran-70, and Ficoll-70 (Sigma-Aldrich, St. Louis, MO, USA) were used without additional
purification. The absorbance of protein samples did not exceed 0.15 in all our experiments unless
otherwise indicated. Taking into account the extinction coefficient ε280 = 31,519 M−1 ·sm−1 [19],
this means that protein concentration was not higher than 5 µM. Measurements were conducted in
100 mM Na-phosphate-buffered solution at pH 8.0 in the absence or presence of different concentrations
of crowding agents.
4.2. Gene Expression and Protein Purification
Construction of the plasmid (pET-28a(+)-sfGFP) coding for the poly-histidine tag containing
super-folder GFP (sfGFP, [10]) was conducted following the previously described protocols [49].
Int. J. Mol. Sci. 2016, 17, 1805
11 of 14
Escherichia coli BL21 (DE3) cells (Invitrogen, Waltham, MA, USA) were used for transformation, and
the sfGFP expression was induced by adding 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG;
Fluka, Buchs, Switzerland) with subsequent incubation for 24 h at 23 ◦ C. His-GraviTrap columns
(GE Healthcare, Little Chalfont, UK) packed with Ni+ -agarose were used for the purification of the
recombinant protein. The 15% polyacrylamide gel electrophoresis in the presence of sodium dodecyl
sulfate SDS-PAGE [50] revealed that purity of the sfGFP was at least 95%.
4.3. Spectrophotometric Experiments
An EPS-3T spectrophotometer (Hitachi, Tokyo, Japan) with microcells 101.016-QS 5 × 5 mm
(Hellma, Mulheim, Germany) was used for the absorption measurements, all of which were performed
at room temperature.
4.4. Fluorescence Spectroscopy
A Cary Eclipse spectrofluorometer with microcells (10 × 10 mm; Agilent Technologies, Mulgrave,
Victoria, Australia) equipped with a thermostat was used for conducting fluorescence experiments at
the constant temperature of 23 ◦ C. A recently proposed approach was used for the correction of the
fluorescence intensity for the primary inner filter effect [51,52].
To minimize the contribution of tyrosine residues in the bulk protein fluorescence, the long-wave
absorption spectrum edge (λex = 295 nm) was used for excitation of the protein tryptophan fluorescence.
The effects of environmental factors on the position and shape of the fluorescence spectra were analyzed
using the A = I320 /I365 parameter, with I320 and I365 being the fluorescence emission intensities at
the wavelengths of 320 and 365 nm, respectively. The fluorescence intensity and the parameter A
values were corrected for the instrument sensitivity. The light scattering was recorded at the coincident
excitation and registration wavelengths (of 295 nm). To analyze the fluorescent properties of the neutral
and anionic forms of the “green” chromophore, the corresponding fluorescence these two forms was
exited at 390 and 490 nm and recorded at 450 and 510 nm, respectively.
The unfolding of the protein was initiated by the manual mixing of a native protein solution (50 µL)
with a buffer solution (450 µL). In addition to the desired concentrations of guanidine thiocyanate
(GTC) the buffer in these experiments contained desired concentrations of a crowding agent. An Abbe
refractometer (LOMO, Saint-Petersburg, Russia) was used to measure the refraction coefficient values
needed for the determination of the GTC concentrations in stock solutions. To initiate sfGFP refolding,
the 50 µL aliquot of the pre-unfolded (in 2.5 M GTC) protein was mixed with 450 µL of buffer
or solutions containing desired concentrations of the denaturant and a crowding agent. Protein
solutions containing a desired denaturant concentration, with or without crowding agent at the desired
concentration, were incubated at 23 ◦ C for 24 h. It is worth noting that thus-measured characteristics of
the protein are quasi-equilibrium parameters [15]. According to the control experiments, the dead-time
in these manual mixing-based unfolding-refolding experiments was about 4 s [53,54].
4.5. Circular Dichroism Measurements
A thermostat-equipped Jasco-810 spectropolarimeter (Jasco, Tokyo, Japan) was used for recording
the protein circular dichroism (CD) spectra with a step size of 0.1 nm. Far-UV (in the range of
250–190 nm), near-UV (in the 320–250 nm range), and visible CD spectra (in the range from 550 to
320 nm) were recorded using the 1-mm (far-UV CD) and 10-mm path length cells (near-UV and visible
CD), respectively. An average of three scans was obtained for of all of the protein CD spectra, which
were corrected by subtracting the CD spectra of the appropriate buffer solution.
5. Conclusions
Data reported in this study suggest that, in addition to the expected excluded volume effects,
crowding agents can also cause some changes in the properties of the solvent. This hypothesis
is supported by the observed increase in the degree of sfGFP aggregation during the protein
Int. J. Mol. Sci. 2016, 17, 1805
12 of 14
unfolding-refolding processes with the increase in the molecular mass and concentration of PEGs.
In fact, it is evident now that the behavior of biomolecules in the conditions of macromolecular
crowding cannot be explained only within the frames of the excluded volume effects and/or direct
interaction between tested biomolecules and crowding agents [28,29,33,34]. The changes in the sfGFP
stability towards the GTC action and the capability of this protein to refold in the presence of different
crowding agents are shown here to be dependent on the type and concentration of the polymer
used as a crowding agent. Furthermore, in the case of PEG, the effects of the crowder is further
influenced by the molecular mass of the polymer. This further supports the idea that, in addition
to the excluded volume, some additional factors are involved in the modulation of the behavior of
sfGFP in the crowded milieu. Accumulated experimental data including results obtained in this
study indicate that crowding agents may affect the solvent properties of aqueous solutions in different
manners, depending on the crowding agent macromolecule structure and the concentration [33,34,55].
These solvent properties are the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and
hydrogen-bond acceptor basicity of aqueous solutions [55]. The diverse impact of different polymers
on the solvent properties of water leads to the unequal changes of the protein properties in the crowded
environment modeled by these polymers. Therefore, restructuring of aqueous media might contribute
greatly to the biological processes in a crowded milieu.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/17/11/1805/s1.
Acknowledgments: This work was supported by a grant from Russian Science Foundation RSCF No 14-24-00131.
Author Contributions: Olesya V. Stepanenko and Olga V. Stepanenko collected and analyzed data, contributed
to discussion, and wrote the manuscript. Olesya V. Stepanenko, Irina M. Kuznetsova, Vladimir N. Uversky,
and Konstantin K. Turoverov conceived the idea, supervised the project, contributed to discussion, and reviewed/
edited manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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